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Microbiologically Induced Corrosion on Tin Bronze samples simulating uncommon Archaeological corrosion

G. Ghiara1-2, P. Piccardo1-2, M. Stauder3 1Department of Chemistry and Industrial Chemistry (DCCI), University of Genoa

Via Dodecaneso 31, 16146 Genova (ITALY) 2Consorzio Interuniversitario Nazionale per la Scienza e la Tecnologia dei Materiali

(INSTM), Via G. Giusti 9, 50121 Firenze (ITALY) 3Department of Earth, Environment and Life Sciences (DISTAV), University of Genoa

Corso Europa 26, 16132 Genova (ITALY) Email: giorgia.ghiara@gmail.com

Summary

Microorganisms may develop a tolerance towards copper ions and influence the corrosion process – so called Microbiologically Induced or Influenced Corrosion (MIC). The most com-monly studied alloys connected to microbial colonization are: copper-nickel, brass and alu-minum brass. Microbial attack on tin-bronze materials has been discussed recently in the field of cultural heritage. Corrosion of copper based objects involving foreign agents such as microorganisms might involve additionally other than common morphological features. The detection of the unique morphology defined ‘tentacle like’ and elemental composition in cross section corrosion features required the classification of a new MIC mechanism. The study of MIC on archaeological tin-bronzes furthermore might provide due to their long exposure to the environment and their soon stabilized bacterial attack the key to the solution of how to stabilize or limit MIC on copper alloy based objects. This study focuses on the reproduction of such morphological aspects by experimental ses-sions. Analyses were carried out with Light Optical Microscopy (OM), Scanning Electron Mi-croscopy coupled with Energy Dispersive X-Ray Spectroscopy (SEM-EDXS) and Micro-Raman Spectroscopy (μRS).

1 Introduction

Corrosion mechanisms for metallic objects follow general rules well explicable by the

influence of some predominant factors as environmental, physical or chemical ones:

pH, temperature, presence of oxygen or reactive ions in solution (such as Cl-, SO42-,

NO3-, CO3

2-, or others) [1–6]. Each morphological feature suggests a specific corro-

sion mechanism which obeys thermodynamic and electrochemical laws [7–10].

The most commonly noted corrosion products are copper- and tin oxides as well as

copper nitrates, -sulfates and -carbonates which appear in cross sections as pitting,

intergranular or crevice corrosion [11,12].

It is well known that copper acts as a biocide for any type of organism; but it is also

true that some of them – mostly microorganisms – may develop a tolerance towards

copper ions. In this case corrosion mechanisms are influenced by microbial agents –

so called Microbiologically Induced or Influenced Corrosion (MIC) [13–15]. Research

on copper based alloys linked to microbial colonization is usually connected to sani-

tary problems: seawater piping system exposed to estuarine and seawater or weld

and heat affected zones usually provide ideal conditions for the colonization of differ-

ent kinds of bacteria [16]. Few tin-bronze materials under microbial attack have been

analysed, being MIC only studied on few copper based alloys: copper- nickel, brass

and aluminum brass [14].

The case of archaeological objects is quite peculiar since defined corrosion mecha-

nisms can be altered by a combination of factors which act as competitive agents in

the reaction [17–19]. According to their long deposition in soil or water context, which

might be up to several thousand years and change significantly during time, the for-

mation of corrosion products on archaeological tin-bronze vary according to nature

and composition of the environment they are exposed to [20–22]. Indeed, archaeo-

logical corrosion might show besides common morphological features further ones,

particularly when ‘foreign’ factors – agents such as microorganisms – are involved

[23]. The detection of a specific, penetrating corrosion feature, called ’tentacle-like’ by

its peculiar shape when observed in cross section, was recently noted on several tin-

bronze objects dated to the European Late Bronze Age (c. 1200-1000 BC) by Pic-

cardo et al. [24]. The morphology as well as the composition differed so much from

classical models that a new corrosion type had to be defined. Moreover, some mor-

phological and chemical aspects suggested the intervention of corrosive agents nev-

er considered before. This type of corrosion penetrated the metallic matrix forming 1-

1.5 μm wide tunnel shaped structures, not following any microstructural features as

grain- or twinning boundaries, or slip bands. Additionally, inside these so-called ‘ten-

tacles’ anomalous concentration of silicon, phosphor and even aluminum, manga-

nese and iron were detected. This observation lead to the hypothesis that the pres-

ence of these elements in the described corrosion structure can be related to the

presence of MIC [25]. It is also worth noting that archaeological bronzes with such

features cannot be analysed by standard MIC techniques but alternative approaches

– such as metallographic ones - have to be applied. In our specific case archaeologi-

cal bronzes are subjected to a long term corrosion process and it is not possible to

determine if their corrosive action is related to a specific timespan and whether other

parameters are involved or not.

The study focuses on the reproduction of such features by experimental sessions

using a specifically selected strain of Pseudomonas fluorescens following literature

data [15]. The choice of this strain has to be related to its relative tolerance towards

copper ions and their growth on copper samples or in presence of copper ions as it is

well explained in many research papers [16,26,27]. In order to verify the correlations

between archaeological and laboratory morphologies and corrosion products, anal-

yses were performed using Light Optical Microscopy (OM), Scanning Electron Mi-

croscopy coupled with Energy Dispersive X-Ray Spectroscopy (SEM-EDS) and Mi-

croRaman Spectroscopy (μRS). In particular the study was carried out in order to: 1. recreate tentacle like corrosion morphologies by selecting the adequate

parameters for microbiological growth and verify the effects of metallurgical

structure on such corrosion;

2. chemically characterise the corrosion products in order to verify the corro-

sive capabilities of such microorganisms on tin-bronzes;

3. reproduce the exact corrosion mechanism observed for archaeological ob-

jects.

2 Materials and methods

The study was carried out following the work of some of the authors [24] on different

types of corrosion of archaeological bronze objects, which were found in different

environment (e.g. soil or water context) at different archaeological sites all across

Europe. A specific corrosion morphology discussed in the previous work was select-

ed in order to minimize the influence of the external parameters: Type II corrosion

morphology described in the paper. This morphology was defined by Piccardo et al.

[24] and occurs on objects which were found in waterlogged environments. It con-

sists of a tangled structure with tunnels that are not much widened by the secondary

corrosion.

2.1 Pseudomonas growth

An animal isolate of P. fluorescens, kindly provided by Prof. Marchese (University of

Genova) was used in this study. Pure cultures of P. fluorescens were grown at 32° C in sterile artificial tap water (ATW) amended with 0.06% peptone, with continuous shaking. The mineral base composition of AWT was MgSO4 (40 mg L−1), MgCl2 (60 mg L−1),KNO3 (25 mg L−1), CaCl2 (110 mg L−1), Na2CO3 (560 mg L−1) and NaNO3 (20 mg L−1) in distilled water; the solution was autoclaved and the pH adjusted to 7.6 [22,26].

After overnight incubation the cells were washed twice with AWT and resuspended in

AWT at the final concentration of 1x107 CFU/ml (colony forming unit/ml).

2.2 Experimental setup

Tin-bronze cubes of 1 x 2 x 0.5 cm size were produced. They were made of c. 88

wt% Cu and 12 wt% Sn. Table 1 resumes the elemental composition of tin bronze

alloy used for the experiments.

Table 1: average alloy composition in wt% of the tin-bronze ingot (analyses carried out by

SEM-EDS).

Alloy number UNI EN

Al Si P S Fe Ni Cu Zn Sn Sb Pb

CuSn12 CC483K 1982 0.2 0.2 0.1 0.1 0.1 0.1 87.8 0.3 11.2 0.1 1.2

In order to reproduce the detected microstructure of the archaeological samples, four

sample sets were produced. Set I consists of as-cast samples (‘raw’). The procedure

for treated samples (set II-IV) consisted of three steps (I, II, III) of mechanical treat-

ments (cold hammering) followed by annealing at 620°C (see table 2). Last treat-

ment for each step was cold hammering in order to enhance crystal defects.

Table 2: Specimens used for the experimentation. One samples for each set was treated as

our ‘blank’ in order to identify artificial water corrosion and differentiate it from the MIC.

Set Treatment Number of steps Number of samples

Hammering/ Annealing

Blank MIC

I raw 0 1 2

II Step I 1 1 2

III Step II 2 1 2

IV Step III 3 1 2

All samples were accurately shaped and polished using metallographic emery paper

(SiC) up to 1000 grit and subsequently cleaned with distilled water and ethanol in

order to remove corrosion traces and residues of the polishing operation. All samples

were then autoclaved and immersed in bacterial suspension in order to verify the mi-

crobiological influence on corrosion. An exception was made for samples immersed

in pure artificial water which consisted in our ‘blank’ samples. The corroded speci-

mens were withdrawn after 2 months exposition time and washed gently with distilled

water in order to eliminate adhered bacteria. Figure 1 resumes the experimental pro-

cedure used.

Figure 1: experimental setup: a) samples were polished; b) autoclaved; c) immersed either in bacteri-

al solution or artificial water.

2.3 Protocol of Investigation

Morphological observations were carried out using a Light Optical Microscope (OM)

(6x, 12x, 25x, 50x, 100x magnification), in order to differentiate the corrosion prod-

ucts by their morphological shape and colour. Afterwards, selected samples were

embedded in hot resin and polished with SiC paper (up to grit 1000) and then by di-

amond suspension up to 250 nm of granulometry.

Scanning Electron Microscopy (SEM) analysis was carried out using a ZEISS EVO

40. All SEM micrographs showed both crystalline growth on the surface and the cor-

rosion morphologies in cross sections and they were obtained using an acceleration

voltage of 20 KV and a detector for secondary electrons (SE). Energy-dispersive X-

ray spectroscopy (EDS) was performed using the following parameters: implement-

ing time of 50 s; ZAF 5 correction with Pentafet detector sensitive to light elements.

Micro-Raman analyses were performed on the surface of the samples using a Ren-

ishaw System 2000 spectrometer with Leica Optical microscope with a 20x long focal

and equipped with a Peltier-cooled CCD detector. A Red He-Ne laser excitation

source (632.8 nm wavelength, full power 4 mW) was used with laser power reduced

at 10% and 25% transmission filter in order to avoid sample damages or fluores-

cence effects. Spectra were recorded from 4000 to 100 cm-1. Integration time was 10

seconds and accumulations varied from 1 to 9. Measurements were repeated on dif-

ferent points. Spectra obtained were compared to data published in literature and

RRUFF Project database (www.rruff.info).

3 Results and Discussion

3.1. Analysis on the surface

Analysis by OM revealed a variety of colours which are usually associated to different

corrosion products that form on the surface of the samples. Each specimen shows a

different morphology depending on the mechanical treatment applied. On the sam-

ples of set I, a very thin surface patina layer was noted for both blank and MIC sam-

ples (figure 2a,b), assuming a slower corrosion rate. Instead, a thicker patina layer

was observed on specimens that were mechanically and thermally treated, which is

coherent with general corrosion processes (figure 2c-g). Black areas of different size

were noted on the surface of all samples exposed to bacterial culture and on ‘blank’

samples of set II, III, IV (figure 2b-g).

Figure 2: OM micrographs at different magnification of selected samples. First line from left to right: a)

‘blank’ set I; b) set I; c) blank’ set II; d) set II; second line from left to right: e) ‘blank’ set III; f) set III; g)

set IV. Marker lines indicate mm.

SEM observations allowed us to distinguish the nature of the patinas which differs on

all the samples. A partially adherent compact layer is always visible (figure 3a-d).

These areas appear not fully crystallised and round shaped structures are noted in-

side the compact matrix.

a b c d

e f g

a b c d

e f g

Figure 3: SEM-SE micrographs at different magnification of selected samples. First line from left to

right: a) ‘blank’ set I, 1000x; b) set I, 2500x; c) blank’ set II, 1500x; d) set III, 3000x; second line from

left to right: e) set II, 3000x; f) set III, 9000x; g) set IV, 3500x.

EDXS and Raman indicated a mixture of copper (I) and tin (IV) oxide (respectively

Cuprite or Cu2O and Cassiterite or SnO2) in which Cassiterite is present at different

crystallisation degrees [28] (figure 4a-c). EDXS also allowed us to identify calcium

oxides which are present onto the patina as a result of precipitation from the initial

solution. It is also worth noticing that all treated samples exhibit small areas with a

dark black patina. It consists of copper (II) oxide Tenorite or CuO (see figure 4d). Its

presence is connected to the high temperature oxidation during annealing procedure.

Figure 4: Raman spectra of copper and tin oxides on representative samples: a) nanoscopic Cassiter-

ite; b) Cuprite and Cassiterite; c) Cuprite, Tenorite and Cassiterite; d) Tenorite. Each spectrum was

recorded with the following parameters: acquisition time: 10 s.; accumulations number: 4; power: 25%

transmittance.

The blackish-grey areas appears on the surface of all samples of set II, III and IV ex-

posed to Pseudomonas fluorescens. Differences in morphology are noticed (figure

3e-g). Set II and III show a non-homogeneous layer which is very adherent to the

metallic surface and globular shaped structures are observed (figure 5).

Figure 5: SEM-SE 1500x micrograph of the blackish grey area on a representative sample from set

III. Spectra results are shown in table 3.

EDXS analysis on some areas allowed us to identify copper sulphides: Covellite type

or CuS (see table 3).

Table 3: results in at% from the spectra measured on the corrosion as shown in Figure 5.

Site of interest

O Al Si P S Cl Cu Sn

Spectrum 1 20.3 22 57.7

Spectrum 2 17.4 17.5 61.1 4

Spectrum 3 32.2 0.2 0.1 0.4 13.2 0.1 48.6 4.9

Samples of set IV reveal a well crystallised patina which is consistent with the detec-

tion of a different sulphur based compound. The substrate is made of acicular crys-

tals which distribute inside cracks and holes of the sample. Microanalysis and Raman

confirmed the presence of the copper sulphate Antlerite (or Cu3(SO4)(OH)4) (see fig-

ure 6).

Figure 6: First line: elemental mapping of a representative sample from set IV. From left to right: SEM-

BSE micrograph with O, S, Cu and Sn distribution in the analysed area. Second line: raman vibrational

bands of antlerite (and non-attributed peaks=*) compared to antlerite reference from RRUFF and spot

of analysis.

3.2 Analysis on cross section

Selected samples were mounted in resin and polished in order to characterise their

cross sections. The corrosion features were then morphologically and chemically

characterised and their penetration into the metal was identified. Figure 7 resumes

the corrosion morphology observed.

a b c d

O S Cu Sn 70μm

Figure 7: SEM-SE micrographs at different magnification of selected samples. From left to right: a)

set II, 4000x; b) set III, 8500x; c) set III, 9000x; d) set III, 10000x. The tentacle like feature is visible.

High magnifications allowed us to observe a rather visible spot with starting ‘tentacle

like’ penetration which forms directly under the microbial attack. High concentrations

of sulphur and copper were especially detected on the upper corrosion layer and ra-

ther uncommon elements such as Si, Al and P are noted at the interface corro-

sion/metallic matrix (see table 4). Such elements are minor alloying elements (see

table 1) absent in the solution. Their higher concentration in the MIC spots has there-

fore to be directly connected to the action of bacteria as the presence of Si-rich com-

pounds.

Table 4: elemental analysis in wt% at the interface corrosion/metallic matrix under CuS pati-

na.

Site of interest

O Al Si P S Cl Cu Sn Pb

Spectrum 1 18.2 2 3.9 0.1 3.7 0.6 54.7 16 0.8

Spectrum 3 22.9 1.8 0.2 0.2 0.5 0.4 35.9 36.3 1.9

Spectrum 4 12.9 1.4 0.3 0.2 0.1 0.3 63.1 20.8 1

Spectrum 5 12.2 0.9 0.3 0.6 4.5 71.5 8.4 0.8

From these first experiments we can note that the detection of sulfur base com-

pounds as Chovellite and Antlerite (respectively CuS and Cu3(SO4)(OH)4) indicate

the active influence of bacterial culture on the corrosion of many samples [14,25].

Corrosion processes influenced by microbial activity was noted mainly on mechani-

cally treated specimens: it is therefore possible to assume a preferential corrosion

induced by bacterial cultures on samples which underwent a mechanical and thermal

treatment due to the higher number of crystal defects (e.g. grain boundaries)., sug-

gesting their influence on the corrosion process. In particular copper sulphates occur

inside cavities and holes created by the mechanical treatment, indicating a preferen-

tial corrosion site suitable for microbial attack.. Also tentacle like structures are ob-

served inside the corrosion layers which are dependent upon microbial colonisation.

Further researches are anyway needed to better characterize corrosion process in-

duced by such microorganisms.

4 Conclusions

First results from the reproduction of tentacle like corrosion features by experimental

sessions are presented. Corrosion processes influenced by microbial activity was

noted mainly on mechanically treated specimens, suggesting the influence of crystal

defects on such type of corrosion process. Copper sulphides and sulphates are de-

tected as well as uncommon amounts of elements such as silicon, aluminum and

phosphorous. Accordingly further researches are needed in order to acquire more

knowledge on this corrosion process.

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